The objective of the proposed research will be to provide a biochemical basis for the rational synthetic design of potentially active chemotherapeutic pyrimidine, pyrimidine-like heterocycles and the corresponding nucleosides. Evaluation of in vitro cytotoxicity in L-1210 and H.Ep. #2 cell cultures will be accomplished for all compounds synthesized on our NIH grants. We propose to obtain a preliminary indication of the overall metabolic effects of cytotoxic compounds by testing the reversibility of growth inhibition and cell killing by naturally occurring nucleosides. On the basis of these in vitro studies and the in vivo antitumor evaluation by DCT, NCI, analogs of interest will be selected for further investigation. Studies on the biochemical effects of these analogs will include incorporation of 14C-orotic acid into acid soluble and RNA nucleotides, and release of 14CO2 from 14C-orotic acid in analog-treated cell suspensions. Elucidation of the structural and physicochemical requirements for substrate activity of uridine-cytidine kinase, pyrimidine nucleoside phosphorylase, and cytidine deaminase will be studied, using the series of related compounds available from the synthetic program. This information should aid in the synthetic design of analogs to be activated by cellular metabolic reactions, and to avoid inactivation. Also, the knowledge of which analog metabolites may be present in the cell, together with the results from the studies described above, should provide a basis for proposing possible mechanisms of action for cytotoxic analogs.